The contractile proteins actin and myosin are widely types of eukaryotic cells, distributed in different types of eukaryotic cells. These proteins participate in a variety of cellular functions ranging from the obvious, cell motility, to cell division, cellular secretory processes and immunological reactions at the cell surface. Little is known about the molecular sequence of events, known as excitation-contraction (E-C) coupling, which regulates the interaction of actin and myosin. The overall objective of this project is to learn the nature of these events. Our specific objectives will be to: (1) Use myoepithelial (ME) cells as a model system to study E-C coupling; (2) Determine the relationship between oxytocin (OT) receptor occupancy at the ME cell surface and phosphorylation of myosin by myosin light chain kinase (MLCK); (3) Determine the temporal relationship between OT binding and myosin phosphorylation to see which of the two is rate limiting in E-C coupling; (4) Determine the mode of regulation of MLCK, in particular, whether it is controlled by changes in Ca2 ion concentration. We will isolate ME cells from lactating and involuted mammary glands using collagenase cell dispersion techniques (ME cells do not regress as do secretory cells). The cells will be incubated in radioactive phosphate and the extent and rate of labelling of intracellular ATP pool(s) will be measured using high pressure liquid chromatography. The kinetics of OT binding will be determined under conditions where OT is known to cause contraction of ME cells and under conditions where there is some 'slippage" of the E-C coupling mechanism. The myosin content of the cells will be determined by a combination of radiochemical and electrophoretic procedures. When we have determined the above parameters we will measure intracellular phosphorylation of myosin under various conditions of OT receptor occupancy. Myosin light chain kinase will be isolated from ME cell cytosol and its control properties studied using myosin light chain from smooth muscle as substrate.